商品编号


I5HD-200



品牌


MCLab



公司


MCLAB.Inc



公司分类


PCR Polymerases



Description

1,250 Units,2.5 U/μl


Size

1 Ea

商品信息

I-5 Hi-Fi DNA HotStart Polymerase is a high-fidelity and?fast DNA polymerase.




General Description
I-5 Hi-Fi DNA HotStart Polymerase is an ultra-fast and high-fidelity DNA polymerase. It provides robust amplification of difficult templates including plasmids, BACS, genomic DNA, and lam
BD
a DNA. It allows the amplification of up to 8kb human genomic DNA and up to 21kb lam
BD
a DNA. It has an extension speed of 1 kb / 10-15 seconds depending on template type. This allows users to save time by speeding up PCR reactions and provides higher fidelity than
Taq
or
Pfu
. The enzyme contains a HotStart mechanism that inactivates the enzyme until it is heated. This allows users to setup PCR reactions at room temperature without worrying about primer dimers or non-specific preamplification.
Feature
? Fast – 4X faster than hot start
Taq
? Robust – high inhibitor tolerance
? High yields – high efficiency
? Long PCR products


Source
E. coli


Applications
Hot Start PCR
Routine PCR
Fast PCR
High throughput PCR
Genotyping


Supplied with
5X Reaction Buffer (1ml, 5X MgCl
2?
included?)

50mM MgCl
2
(1ml) (for optimization)


Supplied in (buffer description)
20mM Tris-HCl, 100mM KCl, 1mM DTT, 0.1mM EDTA, 200ug/ml BSA, 50% glycerol, 1X st
ABI
lizer, pH 8.0 @ 25 C


Storage Condition
-20?C


Unit Definition
One unit is the amount of enzyme that incorporates 5 nmol of dNTPs into acid insoluble material in 15 minutes at 72°C.


Reference
Frey, B. and Suppman, B. (1995). BioChemica. 2, 34-35.
Chester, N. and Marshak, D.R. (1993). Analytical Biochemistry. 209, 284-290.


Shipping Condition
dry ice


User protocol

?

?

25 ?l Reaction

50 ?l Reaction

Final Concentration

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Instructions

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25 ?l Reaction

50 ?l Reaction

Final Concentration

I-5 5X Buffer

5 ul

10 ul

1X (see notes)

10 ?M Primer A

1 ?l

2 ?l

400 ?M

10 ?M Primer B

1 ?l

2 ?l

400 ?M

Template DNA

as needed

as needed

see notes

50mM MgCl
2

as needed

as needed

see notes

I-5 Enzyme

0.5 - 1 ul

1 - 2 ul

?

Water (ddH
2
O)

up to 25 ?l

up to 50 ?l

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Thermo
cycling Conditions

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3 Step PCR

?

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Step

Temperature

Time

?

Initial Denaturation

98°C

2 minutes

?

Denaturation

98°C

10 seconds

25-35 cycles

Annealing

55°C - 68°C

10-15 seconds

?

Extension

72°C

10-15 seconds / kb

?

Final Extension

72°C

1-5 minutes

?

?

4°C

Hold

?

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2 Step PCR (see notes)

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Step

Temperature

Time

?

Initial Denaturation

98°C

2 minutes

?

Denaturation

98°C

10-15 seconds

25-35 cycles

Combined Annealing & Extension

72°C

15-30 seconds / kb

?

Final Extension

72°C

1-5 minutes

?

?

4°C

Hold

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Notes

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Recommended DNA Template addition

?

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Genomic DNA

50-250 ng

?

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Plasmid DNA

1pg-10ng

?

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Viral DNA

1pg-10ng

?

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Mg
2+

?

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The final concentration of Mg
2+
in 1X I-5 PCR Master Mix is 1.5 mM

?

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Add additional Mg
2+
as needed in 0.5 mM increments

?

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Suggested final Mg
2+
concentration ranges from 2 mM to 4 mM

?

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2 Step PCR

?

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Use 2 Step PCR is recommended when the primer T
m
values are >68°C

?

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PCR Product / Cloning

?

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The 2X I-5 PCR Master Mix results in PCR product with blunt ends

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Blunt-end cloning is recommended after PCR with this product

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For T/A-cloning, the PCR product should be purified before A-addition

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